Adequate neovascularization is a prerequisite for progressive tumor growth (1). Neoangiogenesis is required in particular for maintaining expansive tumor growth, since only sufficient oxygenation will ensure the supply with nutrients to and removal of tumor degradation products from the tumor.
In the prior art directed to tumor treatment antivascular therapeutic strategies have been developed, which are aimed at destruction of the tumor blood vessels and associated tumor infarction, in addition to anti-angiogenic therapeutic strategies, which attack the complex process of growth and differentiation of blood vessels.
A precondition for these strategies is identification of target structures in the vascular endothelium of the tumor that do not occur on resting endothelial cells in normal tissue. These specific target structures could be utilized in order to apply cytostatics or certain toxins to the vascular endothelial cells of the tumor to a lesser extent to the tumor cells themselves. Target structures that can be used for this purpose are bFGF (basic fibroblast growth factor), VEGF (vascular endothelial growth factor) and VEGFR-2 (VEGF receptor 2), endoglin, endosialin, a fibronectin isoform (ED-B domains), the integrins αvβ3, αvβ5, α1β1, and α1β2, aminopeptidase N, NG2 proteoglycan and the matrix metalloproteinases 2 and 9 (MMP 2 and 9) (2-13). For example, Arap et al. (8) coupled peptides that bind alpha1-integrins specifically, to an active substance that was being used in the state of the art for chemotherapy (doxorubicin). It was demonstrated in an animal model that the antineoplastic effect of doxorubicin could be improved by coupling to the peptides.
An alternative antivascular therapeutic approach comprises selective activation of blood clotting in tumor vessels, in order to induce tumor necrosis. For example, a bispecific F(ab′)2 antibody fragment was produced, which is directed against truncated tissue factor (tTF) and an MHC class II antigen. After experimental induction of the antigen in tumor endothelial cells, an antivascular therapy could be demonstrated by administering the antibody in a murine neuroblastoma model (14). In a second study by the same team, an immunoconjugate was used, which couples tTF selectively to a naturally occurring marker of the tumor vessel endothelium, VCAM-1 (vascular cell adhesion molecule-1) (15).
In a very similar approach, an antibody fragment (scFv), which is specific for the oncofetal ED-B domain, was fused with tTF. The fusion proteins generated, scFv-tTF, led to a complete and selective infarction in various tumors in the mouse model (16).
Alternatively, tTF was coupled to an inhibitor of the prostate-specific membrane antigen (17). This fusion protein induced selective infarction necrosis in a rat prostate model after intravenous administration. Administering this fusion protein in combination with a cytotoxic substance (doxorubicin) at low dose resulted in massive tumor regression and even complete tumor eradication (17). Other tTF fusion proteins, consisting of antibody fragments against VEGFR (VEGF receptor), endoglin and VCAM-1, have been described recently (18).
However, the molecules produced for antivascular tumor therapy in the state of the art have drawbacks. In particular it has to be assumed that these molecules are immunogenic owing to their size. Treatment of mammals with these molecules will therefore trigger an immune reaction against the molecules, so that repeated administration of the molecules becomes impossible.
The size of the coupling partner, by means of which the peptide portion, which can activate blood clotting, is to be directed onto the tumor tissue, may further cause steric hindrance to a formation of the macromolecular factor VIIa/FX enzyme-substrate complex, which is important for blood clotting. Formation of the complex can also be hampered when the peptide capable of activating blood clotting has an altered conformation owing to the relatively large fusion partners.
In the state of the art (WO 03/035688), fusion polypeptides are also known wherein a selective binding domain, e.g. a domain of fibronectin that binds to integrins, e.g. which comprises RGD peptides, or the D-β-E dipeptide, which binds to PSMA (prostate-specific membrane antigen), is coupled to the N-terminus of a tissue factor polypeptide. Although an amidolytic and proteolytic effect was demonstrated in vitro, the constructs, even in combination with factor VIIa, only displayed extremely weak anti-tumor effect in vivo. The animals only survived longer in combination with doxycycline.
Hu et al. (46) describe various fusion proteins and use thereof for the production of thromboses in tumor vessels, including a fusion protein from an oligopeptide with 9 amino acids, containing the RGD sequence, which was coupled to the truncated form of the tissue factor. Again, the RGD peptides were linked to the N-terminus of tTF to obtain RGD-tTF. Functional analysis showed that the fusion protein containing RGD did not produce any significant inhibition of tumor growth.
The constructs known in the state of the art were thus constructed in such a manner that the selective binding domain was linked to the N-terminus of the tissue factor polypeptide. It was even emphasized that this structure must be chosen because the N-terminus, on the basis of structural models, was considered to be an especially favorable site for linkage, which would not inhibit the initiation of thrombosis.